To establish a dual RT-qPCR detection method for porcine rotavirus A ( PoRVA) and seneca virus A ( SVA) and conduct preliminary applications. Methods By comparing the sequences of PoRVA and SVA strains, conserved regions of the PoRVA NSP3 gene and SVA VP1 gene were selected to design primers and probes, respectively, and a dual RT-qPCR detection method for PoRVA and SVA was established. Subsequently, the specificity and sensitivity of the method was confirmed.Recombinant plasmid standards with concentrations ranging from 10 6 to 10 3 copies/ μL were serially diluted 10 - fold. Each concentration was tested in triplicate and the tests were repeated three times.Intra- and inter-assay coefficients of variation ( CV) were calculated to evaluate the repeatability and stability of the method. The established dual RT-qPCR method was compared with the detection method recommended by national standard “ GB / T 34756 - 2017” and industry standard “ SN / T 5486 - 2022” .Additionally, 15 pig fecal samples and 35 pig anal swab samples were tested to evaluate the practical applicability of the method. Results The established dual RT-qPCR method showed a linear relationship between Ct values and plasmid concentrations in the range of 109-101copies/ μL. The standard curve for PoRVA had a slope of -3. 085, an R 2 value of 0. 993, and an amplification efficiency of 110. 956%. The standard curve for SVA had a slope of -3. 085, an R 2 value of 0. 993, and an amplification efficiency of 110. 935%. The minimum detectable concentrations for both PoRVA and SVA were 101 copies/ μL. No cross-reactivity was observed with other seven common swine pathogens. Repeated testing of plasmid standards at concentrations of 106 to 103 copies/ μL showed intra-assay CVs of less than 0. 15% and inter assay CVs of less than 1. 50%. The established dual RT-qPCR method and the national standard and industry standard method were used to detect the positive rate of 50 samples. The result showed that the positive rate of PoRVA and SVA were 100% (50 / 50) and 66% (33 / 50) for the dual RT-qPCR method, respectively. The positive rate of PoRVA and SVA were 100% (50 / 50) and 24% ( 12 / 50) for “GB / T 34756- 2017” and “ SN / T 5486 - 2022” method, respectively. The positive concordance rate reached 100%. Conclusion The dual RT-qPCR detection method established in this study can effectively detect PoRVA and SVA, providing technical support for disease detection and routine monitoring in swine populations.

Porcine rotavirus A, Seneca virus A, establishment and application of a duplex RT-qPCR detection method

目的 建立 A 群轮状病毒( PoRVA) 和塞内卡病毒( SVA) 双重 RT-qPCR 检测方法,并对其进行初步应用。方法 通过比对 A 群轮状病毒和塞内卡病毒毒株序列,选取 PoRVA NSP3 基因、SVA VP1 基因保守区域分别设计引物和探针,建立 PoRVA 和 SVA 双重 RT-qPCR 检测方法。 随后对方法的特异性和敏感性进行检测;取浓度为10 6 ~ 10 3 copies/ μL 10 倍系列稀释的重组质粒标准品,每个浓度做 3 个平行,重复检测 3 次,计算组内和组间变异系数,评估方法的重复性和稳定性;用建立的双重 RT-qPCR 检测方法与《 GB / T34756—2017 猪轮状病毒病病毒RTPCR 检测方法》和《 SN / T 5486—2022 塞内卡病毒病检疫技术规范》的检测方法进行比对,并检测 15 份猪粪便样品和 35 份猪肛拭子样品,评估该方法的实际应用性。 结果 建立的双重 RT-qPCR 方法在 10 9 ~ 10 1 copies/ μL 范围 Ct值与质粒浓度呈线性关系,PoRVA 标准曲线 Slope 为- 3. 085,r 2 值为 0. 993,扩增效率为 110. 956%,SVA 标准曲线Slope 为-3. 085,r 2 值为 0. 993,扩增效率为 110. 935%,可检测到的 PoRVA 和 SVA 最低限均为 10 1 copies/ μL;且与其他 7 种实验猪常见病原不发生交叉反应;重复检测 10 6 ~ 10 3 copies/ μL 质粒标准品,组内变异系数小于 0. 15%,组间变异系数小于 1. 50%。 用建立的双重 RT-qPCR 方法与《 GB / T34756—2017 猪轮状病毒病病毒 RT-PCR 检测方法》和《 SN / T 5486—2022 塞内卡病毒病检疫技术规范》 方法检测 15 份猪粪便样品和 35 份猪肛拭子样品,结果显示,RT-qPCR 检测出 PoRVA 阳性 100% ( 50 / 50) ,SVA 阳性 66% ( 33 / 50) ,《 GB / T34756—2017 猪轮状病毒病病毒RT-PCR 检测方法》检测阳性率 100% ( 50 / 50) ,《 SN / T 5486—2022 塞内卡病毒病检疫技术规范》 检测阳性率 24%(12 / 50) ,阳性符合率达到 100%。 结论 本研究建立的双重 RT-qPCR 检测方法可以有效地检测 PoRVA 和 SVA,可为猪群的疫病检测和日常监测提供技术支撑。

A 群轮状病毒, 塞内卡病毒, 双重 RT-qPCR 检测方法建立及应用